Serotype-specific identification of enterovirus 71 by PCR

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Abstract

Background: Enterovirus 71 and coxsackievirus A16 are closely related genetically and are causative agents of hand foot and mouth disease. Because enterovirus 71 is more often associated with severe neurological disease, there is a need to rapidly discriminate between enterovirus 71 and coxsackievirus A16 during hand, foot, and mouth disease outbreaks. Objectives: Our goal was to develop and evaluate a serotype-specific reverse transcription-polymerase chain reaction (RT/PCR)-based typing method for enterovirus 71. Study design: Two sets of PCR primers were designed to match conserved amino acid intervals of enterovirus 71. One diagnostic primer pair contains deoxyinosine at sites of 4-fold codon degeneracy. A second primer pair was designed for use in sequencing and molecular epidemiology studies. Primer pairs were tested on strains encountered in routine diagnostic samples. Results: Using both sets of primers on a panel of 61 prototype enteroviral strains, both primer pairs gave strong positive signals for only enterovirus 71. These primers amplified all enterovirus 71 isolates tested and discriminated between enterovirus 71 and the most closely related enterovirus, coxsackievirus A16. Conclusions: Our RT-PCR assay can be used for specific identification of enterovirus 71 clinical isolates. Furthermore, the 484-bp product of one primer pair has proven useful in sequencing studies to identify distinct genotypes of enterovirus 71.

Introduction

The two leading agents responsible for large outbreaks of hand, foot, and mouth disease (HFMD), a common, usually benign, rash illness in children, are the enteroviruses coxsackievirus A16 (CA16) and enterovirus 71 (EV71). CA16 and EV71 are closely related genetically, but EV71 is also associated with severe neurologic disease, such as encephalitis, meningitis, cranial nerve palsies, Guillain–Barre syndrome, and poliomyelitis-like syndrome much more frequently than is CA16 (reviewed in Chumakov et al., 1979, Gilbert et al., 1988, Alexander et al., 1994, Landry et al., 1995). Two recent EV71 disease outbreaks in Asia (Malaysia, 1997, and Taiwan, 1998) have involved thousands of HFMD cases, some of which were associated with severe neurologic disease and rapid death (WHO, 1997, CDC, 1998, Chang et al., 1998, Lum et al., 1998).

Because of increased public health concerns associated with EV71 and its potential for greater neurovirulence, there is a need for a diagnostic method that can rapidly discriminate between EV71 and CA16 during a large HFMD outbreak. Since the symptoms of EV71- and CA16-associated HFMD are the same, the etiologic diagnosis depends on virus isolation and serotyping. Standard serotyping involves neutralization tests with monospecific antiserum, reagents that are quite limited in their availability. Furthermore, antigenic typing is often hampered by nonneutralizable virus due to aggregation (Blomberg et al., 1974, Schmidt et al., 1974, Nagy et al., 1982). A second typing method has used immunofluorescence techniques utilizing commercially available monoclonal antibodies. End-labeled nucleic acid probes in the VP2 region have also been used in diagnostic tests for EV71 and CA16 (Kitamura et al., 1997).

We report here the development of an EV71-specific RT-PCR assay, using primers in and near the VP1 region. We have developed and evaluated two primer pairs, one that is designed for a rapid-screening, EV71-specific RT-PCR assay and another that is designed for amplification and partial sequencing of VP1 in support of molecular epidemiology studies.

Section snippets

Viruses

The 125 EV71 strains examined in this study include 114 strains previously characterized (Brown et al., 1999, manuscript submitted) and the following additional isolates, with year and state or country of isolation: OK97-2354; CA87-3105; CA88-3104; CA90-3103; MD87-9256; and TAI98-2731; TAI98-2732; TAI98-2733; TAI98-2734; TAI98-2735 and TAI98-2785. The 125 EV71 strains were isolated between 1970 and 1998 at the Centers for Disease Control and Prevention, Atlanta, Georgia, from 25 different

Results

Since enterovirus serotypes are defined by neutralization using immune sera directed against the capsid proteins, nucleotide and amino acid sequences of the capsid region correlate with serotype (Oberste et al., 1999a). In particular, the VP1 -coding region has been shown to specifically correlate with serotype (Oberste et al., 1999a), and this region has been successfully targeted for the development of molecular typing reagents (Kilpatrick et al., 1996, Kilpatrick et al., 1998, Oberste et

Discussion

Awareness of EV71 as an agent of severe neurological disease has increased in the wake of the elimination of poliovirus from the Americas (da Silva et al., 1996) and following fatalities associated with large HFMD outbreaks in Asia (WHO, 1997, CDC, 1998, Chang et al., 1998, Lum et al., 1998). Rapid identification of enteroviruses isolated from paralytic cases in areas no longer endemic for wild poliovirus circulation is essential to the final success of the Global Poliomyelitis Eradication

Acknowledgements

The authors would like to acknowledge laboratories that have provided viruses for screening for EV71. We thank Candra Street for technical assistance.

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    This information was presented, in part, at the annual meeting of the American Society for Virology, July 19, 1997, in Bozeman, Montana, USA (poster P14-3).

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